GSTA is a major glutathione S-transferase gene responsible for 4-hydroxynonenal conjugation in largemouth bass liver.

We have previously shown that largemouth bass (Micropterus salmoides) has a remarkable ability to conjugate 4-hydroxy-2-nonenal (4HNE), a mutagenic and cytotoxic alpha,beta-unsaturated aldehyde produced during the peroxidation of lipids. In addition, we have isolated a glutathione S-transferase cDNA (bass GSTA) that encodes a recombinant protein which is highly active in 4HNE conjugation and structurally similar to plaice (Pleuronectes platessa) GSTA. In the present study, HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with one peak constituting 80% of the total bass liver GST protein. Liquid chromatography mass spectrometry (LC-MS) and electrospray ionization analysis of the major bass GST subunit yielded a molecular weight of 26,396 kDa. Endo-proteinase Lys-C digestion and Edman degradation protein sequencing of this GST peak demonstrated that this protein was encoded by bass GSTA. Analysis of genomic DNA fragments isolated by nested PCR indicated the presence of a GST gene cluster in bass liver that contained GSTA, and was similar to a GST gene cluster characterized by Leaver et al., in plaice. Collectively, our data indicates the presence of a major GST in bass liver involved in the protection against oxidative stress. This GST is part of a gene cluster that may be conserved in certain freshwater and marine fish.

Molecular cloning and characterization of a glutathione S-transferase from largemouth bass (Micropterus salmoides) liver that is involved in the detoxification of 4-hydroxynonenal.

We are currently investigating the role of detoxification pathways in protecting against the sublethal effects of chemicals in largemouth bass (Micropterus salmoides). To this end, previous work in our laboratory indicated a remarkable ability of bass liver glutathione S-transferases (GSTs) to detoxify 4-hydroxynonenal (4HNE), a common mutagenic and cytotoxic alpha,beta-unsaturated aldehyde produced during the peroxidation of lipids. In the current study, we observed that GST-mediated 4HNE conjugation in bass liver follows high efficiency single-enzyme Michaelis-Menten kinetics, suggesting that an individual GST isoform is involved in 4HNE detoxification. Using 5' and 3' rapid amplification of cDNA ends (RACE), a full-length GST cDNA of 957 base pairs (bp) in length, containing an open reading frame of 678 bp and encoding a polypeptide of 225 amino acids, has been cloned. Interestingly, a search of the BLAST protein database revealed the presence of homologous GST proteins in the plaice (Pleuronectes platessa), European flounder (Platichthys flesus) and fathead minnow (Pimephales promelas), but not in other fish species. Furthermore, the bass GST protein exhibited little homology with the mammalian GSTA4 subclass of proteins which rapidly metabolize 4HNE. The recombinant 6 x His-tagged expressed GST protein showed high catalytic activity towards 4HNE, while showing moderate or low activity toward other class specific GST substrates. HPLC-GST subunit analysis, followed by sequencing, demonstrated that the isolated bass liver GST subunit constitutes the major GST protein in bass liver, with a molecular mass of 26.4 kDa. In summary, the presence of a highly expressed GST isozyme in bass and several evolutionarily divergent fish species indicates the conservation of an important and distinct detoxification protein that protects against oxidative damage in certain aquatic organisms.

Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells.

4-hydroxynonenal (4HNE) is a highly mutagenic and cytotoxic alpha,beta-unsaturated aldehyde that can be produced in utero during transplacental exposure to prooxidant compounds. Cellular protection against 4HNE injury is provided by alcohol dehydrogenases (ADH), aldehyde reductases (ALRD), aldehyde dehydrogenases (ALDH), and glutathione S-transferases (GST). In the present study, we examined the comparative detoxification of 4HNE by aldehyde-metabolizing enzymes in a panel of adult and second-trimester prenatal liver tissues and report the toxicological ramifications of ontogenic 4HNE detoxification in vitro. The initial rates of 4HNE oxidation and reduction were two- to fivefold lower in prenatal liver subcellular fractions as compared to adult liver, and the rates of GST conjugation of 4HNE were not detectable in either prenatal or adult cytosolic fractions. GSH-affinity purification of hepatic cytosol yielded detectable and roughly equivalent rates of GST-4HNE conjugation for the two age groups. Consistent with the inefficient oxidative and reductive metabolism of 4HNE in prenatal liver, cytosolic fractions prepared from prenatal liver exhibited a decreased ability to protect against 4HNE-protein adduct formation relative to adults. Prenatal liver hematopoietic stem cells (HSC), which constitute a significant percentage of prenatal liver cell populations, exhibited ALDH activities toward 4HNE, but little reductive or conjugative capacity toward 4HNE through ALRD, ADH, and GST. Cultured HSC exposed to 5 microM 4HNE exhibited a loss in viability and readily formed one or more high molecular weight 4HNE-protein adduct(s). Collectively, our results indicate that second trimester prenatal liver has a lower ability to detoxify 4HNE relative to adults, and that the inefficient detoxification of 4HNE underlies an increased susceptibility to 4HNE injury in sensitive prenatal hepatic cell targets.

Conjugation of 4-hydroxynonenal by largemouth bass (Micropterus salmoides) glutathione S-transferases.

The glutathione S-transferases (GST) are a major group of conjugative enzymes involved in the detoxification of electrophilic compounds and products of oxidative stress. We have previously described the kinetics of hepatic GST conjugation in largemouth bass using a variety of synthetic GST reference substrates. In the present study, we investigated the ability of largemouth bass hepatic GSTs to conjugate 4-hydroxynon-2-enal (4HNE), a mutagenic and cytotoxic alpha-beta-unsaturated aldehyde produced during oxidative injury. Hepatic cytosolic fractions from largemouth bass rapidly catalyzed GSH-dependent 4HNE conjugation, with the rate of GST-4HNE conjugation in bass liver exceeding those of several other mammalian and aquatic species. No apparent sex-related differences in GST-4HNE activity were observed among adult bass. SDS-PAGE and Western blotting analysis of GSH affinity-purified bass liver cytosolic GST revealed the presence of two major GST subunits of approximately 30 and 27 KDa that exhibited slight cross-reactivity when probed with a rat alpha class GST antibody, but not to rat mu, pi or theta class GST. The rapid conjugation of 4HNE by hepatic GST suggests an important role for GSTs in protecting against peroxidation of polyunsaturated fatty acids in bass liver.